Flaviviruses are major human pathogens. They include West Nile, yellow fever and dengue viruses. These viruses result in significant morbidity and mortality each year in temperate and tropical regions of the Earth. West Nile virus has spread throughout the US in the last decade, having caused 4052 recorded infections and 146 deaths in 2006 alone. We intend to continue our structural studies of the viral life cycle in order to elucidate the processes by which the different viral components are assembled first into immature particles and then metamorphose into mature infectious virions. We also plan to continue our structural studies of the path by which these viruses infect their hosts, including the initial host cell recognition, fusion with the host cell plasma membrane initiating endocytosis and, finally, the release of the viral genome into the host cell's cytoplasm. A maturation process that occurs in the final moments before release of infectious virions from a cell is required by most viruses that infect mammals. The immature virions must protect themselves against premature interaction with the cell's own membranes before being released to infect other cells. Last, but not least, we intend to extend our studies of the interaction between antibodies and flaviviruses in order to establish the various mechanisms of neutralization as an aid to the development of vaccines that do not cause antibody-dependent enhancement of infection. Over the past few years we have learned to produce purified dengue (various strains) and West Nile virus in milligram quantities of sufficient quality for structural studies. West Nile virus is especially suitable because of its greater stability, but requires bio-safety level 3 facilities and precautions. These viruses will be used to produce cryo-electron microscopy three-dimensional reconstructions to study immature and mature flaviviruses complexed with various neutralizing antibodies and with cellular receptor molecules. Some of the antibodies inhibit virus maturation or fusion at specific intermediate steps in the viral life cycle. We plan to exploit our recent success in determining the crystal structure of the immature virus'heterodimer ectodomain consisting of the precursor membrane protein (prM) and envelope (E) glycoprotein. This structure has made it possible to build pseudo-atomic models of immature dengue virus and of a low pH intermediate prior to the release of the pr polypeptide and maturation into infectious particles. Mutational and structural studies will now permit determination of the amino acids that control the very large conformational changes that occur when the virus matures into infectious particles. These studies are essential for developing anti-viral and vaccine strategies to establish viable defenses against natural epidemics or bio-terrorist attacks based on using flaviviruses as a weapon. PUBLIC HEALTH RELEVANCE: Flaviviruses, which include West Nile, yellow fever and dengue viruses, are significant human pathogens that give rise to major concerns for human health the World over. Dengue virus alone causes 50 million or more cases of infection worldwide each year, resulting in 24,000 deaths. We propose in-depth studies of the structural changes that occur in the assembly pathway and infection process during the life cycle of flaviviruses, in particular West Nile and dengue viruses. Such information is essential for the development of antiviral drugs and vaccines and for determining the best response in the event of any possible epidemic.